The invention is directed to methods for quantitatively determining the presence and/or amount of a corticosteroid in a solution by forming a corticosteroid-acetate adduct and detecting the adduct using a micromass LC/MS/MS system. These methods accurately detect trace amounts of corticosteroids. Optionally, these methods further detect other drug agents present in concentrations much greater than the corticosteroids to be detected. Preferably, the methods are used to substantially simultaneously detect and determine the amount of dexamethasone and other compounds such as bupivacaine within human plasma.
The quick and accurate determination of drugs within human plasma is of paramount importance in many medical applications. Methods that accurately and quickly determine even trace amounts of a drug in the bloodstream are particularly useful. Moreover, these detection methods become of particular importance in the use of high potency drugs because unwanted side effects are produced when safe dosage levels are exceeded. Potent drugs can include, for example, glucocorticoids which can exacerbate neuronal damage due to hypoxia, ischemia, seizure, and hypoglycemia. Consequently, research has focused on developing drug detection and/or quantification methods that concurrently analyze samples such as plasma for a variety of compounds in differing amounts. This research, however, has been hampered because detection readings can be misinterpreted when the drugs to be detected are structurally similar to other compounds within the sample and/or the drugs are present in minute concentrations within the sample.
The effects of corticosteroids are numerous and widespread. Their diverse effects include: alterations in carbohydrate, protein, and lipid metabolism; maintenance of fluid and electrolyte balance; and preservation of normal function of the cardiovascular system, the immune system, the kidney, skeletal muscle, the endocrine system, and the nervous system. In addition, by mechanisms that are still not fully understood, corticosteroids provide the organism with the capacity to combat stressful circumstances such as noxious stimuli and environmental changes. For example, in the absence of the adrenal cortex, survival is made possible only by maintaining an optimal environment, including adequate and regular feedings, ingestion of relatively large amounts of sodium chloride, and maintenance of an appropriate environmental temperature.
The actions of corticosteroids are related in complex ways to those of other hormones. For example, in the absence of lipolytic hormones, cortisol has virtually no effect on the rate of lipolysis by adipocytes. Likewise, in the absence of glucocorticoids, epinephrine and norepinephrine have only minor effects on lipolysis. Administration of a small dose of a glucocorticoid, however, markedly potentiates the lipolytic action of these amines. These effects of corticosteroids that involve concerted actions with other hormonal regulators are termed permissive and most likely reflect steroid-induced changes in protein synthesis that, in turn, modify tissue responsiveness.
Corticosteroids include glucocorticoids and mineralocorticoids, including, but not limited to, aldosterone, beclomethasone, betamethasone, corticosterone, cortisol, cortisone, dexamethasone, fludrocortisone, flumethasone, hydrocortisone, 6xcex1-methylprednisolone, 6xcex2-methylprednisolone, paramethasone, prednisolone, prednisone, prednylidene, 4-pregnene-20,21-diol-3,11-diol, presesterone, testosterone, triamcinolone, among others.
Two categories of toxic effects result from the therapeutic use of corticosteroids: those resulting from withdrawal of steroid therapy and those resulting from continued used of supraphysiological doses. There are several complications associated with steroid withdrawal, including acute adrenal insufficiency, resulting from too rapid withdrawal of corticosteroids after prolonged therapy, where the hypothalamic-pituitary-adrenal (HPA) axis has been suppressed. Besides the consequences that result from the suppression of the HPA axis, there are a number of other complications that result from prolonged therapy with corticosteroids. These include fluid and electrolyte abnormalities, hypertension, hyperglycemia, increased susceptibility to infection, osteoporosis, myopathy, behavioral disturbances, cataracts, growth arrest, and the characteristic habitus of steroid overdose including fat redistribution, striae, ecchymoses, acne, and hirsutism.
Traditionally, detection methods of corticosteroids have been limited by the inability to detect amounts lower than 100 pg/ml, interference by other compounds including other corticosteroids, extensive and tedious sample preparation, derivatization of samples prior to analysis, or a combination these limitations.
Dexamethasone possesses glucocorticoid activity and is especially used as an anti-inflammatory and anti-allergic drug. Topically, it is employed in the treatment of glucocorticoid-responsive dermatoses. Systemically, dexamethasone decreases the incidence and severity of hearing loss consequent to bacterial meningitis. Its systemic glucocorticoid potency is about 25 times that of cortisone. Dexamethasone is capable of inducing all the usual side effects of adrenal corticoids, except that the mineralocorticoid-like side effects are less pronounced than with cortisone acetate.
Also, glucocorticoids as a group are the most useful class of drugs for treating many eosinophil-related disorders. Glucocorticoids, e.g., dexamethasone, methylprednisolone and hydrocortisone, produce eosinopenia in normal persons, decrease circulating eosinophils in patients with eosinophilia, and reduce eosinophil influx at inflammatory sites (Butterfield et al., Anti-inflammatory Steroid Action: Basic and Clinical Aspects, Schleimer et al., eds., Academic Press, Inc., (1989) at page 151). In 1991, Wallen et al. (J. Immunol., 147, 3940 (1991)) reported the dose-dependent inhibition of IL-5-mediated eosinophil survival by dexamethasone, methylprednisolone and hydrocortisone. Moreover, they disclosed that dexamethasone produced a dose-dependent increase in the EC50 for IL-5-mediated viability enhancement. The relative eosinophil viability inhibitory potencies of the glucocorticoids tested correlated with previously described anti-inflammatory potencies and with the affinities of these agents for the glucocorticoid receptor in the following order: dexamethasone greater than methylprednisolone greater than hydrocortisone.
Bupivacaine was introduced in 1963, and is a widely used amide local anesthetic; its structure is similar to that of lidocaine, except the amine-containing group is a butyl piperidine. It is a potent agent capable of producing prolonged anesthesia. Its long duration of action plus its tendency to provide more sensory than motor block has made it a popular drug for providing prolonged analgesia during labor or postoperative period. By taking advantage of indwelling catheters and continuous infusions, bupivacaine can be used to provide several days of effective analgesia.
Local anesthetics such as bupivacaine block the generation and the conduction of nerve impulses, presumably by increasing the threshold for electrical excitation in the nerve, by slowing the propagation of the nerve impulse, and by reducing the rate of rise of the action potential. In general, the progression of anesthesia is related to the diameter, myelination and conduction velocity of affected nerve fibers. Clinically, the order of loss of nerve function is as follows: (1) pain, (2) temperature, (3) touch, (4) proprioception, and (5) skeletal muscle tone. Systemic absorption of local anesthetics produces effects on the cardiovascular and central nervous systems. At blood concentrations achieved with therapeutic doses, changes in cardiac conduction, excitability, refractoriness, contractility, and peripheral vascular resistance are minimal. However, toxic blood concentrations depress cardiac conduction and excitability, which may lead to atrioventricular block, ventricular arrhythmias and to cardiac arrest, sometimes resulting in fatalities. In addition, myocardial contractility is depressed and peripheral vasodilation occurs, leading to decreased cardiac output and arterial blood pressure.
Bupivacaine is more cardiotoxic than equieffective doses of lidocaine. Clinically, this is manifested by severe ventricular arrhythmias and myocardial depression after inadvertent intravascular administration of large doses of bupivacaine. The enhanced cardiotoxicity of bupivacaine probably is due to multiple factors. Lidocaine and bupivacaine both block cardiac Na+ channels rapidly during systole. However, bupivacaine dissociates much more slowly than does lidocaine during diastole, so a significant fraction of Na+ channels remains blocked at the end of diastole (at physiological heart rates) with bupivacaine. Thus the block by bupivacaine is cumulative and substantially more than would be predicted by its local anesthetic potency. At least a portion of the cardiac toxicity of bupivacaine may be mediated centrally, as direct injection of small quantities of bupivacaine into the medulla can produce malignant ventricular arrhythmias. Bupivacaine induced cardiac toxicity can be very difficult to treat, and its severity is enhanced in the presence of acidosis, hypercarbia, and hypoxemia. Clinical reports and animal research suggest that cardiovascular changes are more likely to occur after unintended intravascular injection of bupivacaine. Therefore incremental dosage is crucial.
As potent drugs such as corticosteroids may induce unwanted side effects if administered in unsafe doses, methods of detecting and/or quantifying these compounds, optionally in the presence of other compounds, accurately and rapidly are still needed. For this purpose, the present invention combines the detection methods of high performance liquid chromatography and mass spectrometry to substantially simultaneously detect relatively small amounts of corticosteroids in the form of adducts in the presence of other drug agents.
LC/MS systems, which combine high performance liquid chromatography (HPLC) and mass spectrometry (MS), are used for several purposes including 1) environmental studies, for example, to evaluate water, soil and waste; 2) food analysis, to identify contaminants and adulterants; 3) pharmaceutical development, to analyze natural and synthetic products; and 4) life sciences, to characterize protein components.
Liquid chromatography is a technique for separating components in a sample mixture. At any given time during separation, some molecules of a component are adsorbed to a stationary solid support, while other molecules are dissolved in a liquid solvent flowing past the solid support. The adsorbed molecules are said to be in a xe2x80x9cstationary phasexe2x80x9d while the dissolved molecules are said to be in a xe2x80x9cmobile phase.xe2x80x9d Separation is based upon the differences of the components"" chemical and/or physical properties. Sample components can differ significantly in their solubility in a given solvent. Specifically, nonpolar components tend to dissolve more readily in organic solvents, while polar components tend to dissolve more readily in water. To accommodate samples with both polar and nonpolar component, reverse-phase gradient-elution liquid chromatography (GELC) provides for a gradual transition of organic solvent to water as the liquid solvent in an LC system.
At equilibrium, the rate at which a component""s molecules in the stationary phase are released to the mobile phase equals the rate at which the same component""s molecules in the mobile phase are adsorbed to the stationary phase. For each component, the ratio of the number of molecules in the stationary phase to the number of molecules in the mobile phase is quantified by a partitioning coefficient. This partitioning coefficient thus corresponds to the average percentage of time the molecules of a component are in the mobile phase. This percentage correlates with the mobility of the component past the solid support. Sample components with different mobilities separate, as they progress past the solid support. With sufficient separation, the components emerge serially in the chromatography effluent.
To complete the analysis of a sample mixture, the eluting components need to be identified and quantified. Various types of detectors, for example, ultra-violet absorption detectors positioned to monitor the ultraviolet absorption characteristics of the effluent, can be used to detect eluting components. Since each component has a characteristic retention time in a chromatographic column, the time of detection is often used for component identification, while the degree of ultraviolet absorption can be used to quantify the component.
However, it is often not possible to identify and quantify sample components dissolved in the chromatography effluent. Some components are not readily detectable, others appear in quantities too small to measure reliably, and others can not be uniquely identified by their retention times. In these situations, and others, a mass spectrometer can be used for sample component identification and quantification.
Mass spectrometry (MS) has long been a widely accepted analytical technique for obtaining qualitative and quantitative information from a sample. MS is commonly used to determine molecular weight, identify chemical structures, and accurately determine the composition of mixtures. A mass spectrometer provides a mass spectrum of a sample component by separating sample subcomponents according to molecular mass and quantifying the number of subcomponent molecules for each molecular mass. Mass spectrometers typically operate by ionizing sample molecules and then sweep-filtering the resulting ions according to their charge-to-mass ratios. To minimize interference with ion movement through the mass filter, mass spectrometers operate under vacuum conditions. MS is becoming increasingly important in biological research to determine the structure of organic molecules based on the fragmentation pattern of ions formed when sample molecules are ionized.
The coupling of mass spectrometers with liquid chromatography systems is a valuable tool for identifying organic compounds. Liquid chromatographic separation systems provide the ability to separate solutions containing mixtures of organic compounds into liquid fractions containing individual compounds. The product of the liquid chromatographic column is an eluant liquid solution of the compound or compounds to be analyzed that is at atmospheric pressure, whereas the mass spectrometer analyzes compounds in a high vacuum system. However, evaporation of the eluant solvent and presentation of the desolvated particles to the mass spectrometer in a suitable form has presented serious difficulties limiting the sensitivity of the mass spectrometer and greatly complicating its efficient operation. Clearly, there is a need for a superior process for detecting trace amounts of cortiocosteriods.
The present invention encompasses a method of detecting a corticosteroid in a sample by adding an internal standard to a sample suspected of containing a corticosteroid; removing, if necessary, interfering compounds from the sample; placing the sample on an HPLC column equilibrated with a NH4OAc:MeOH solution and collecting an eluent; and analyzing the eluent of the HPLC column with a MS, wherein if contained in the sample, the corticosteroid forms an adduct that is detected by the MS. In one embodiment, the corticosteroid is aldosterone, beclomethasone, betamethasone, corticosterone, cortisol, cortisone, dexamethasone, fludrocortisone, flumethasone, hydrocortisone, 6xcex1-methylprednisolone, 6xcex2-methylprednisolone, paramethasone, prednisolone, prednisone, prednylidene, 4-pregnene-20,21-diol-3,11-diol, presesterone, testosterone, triamcinolone, or mixtures thereof, preferably, the corticosteroid is beclomethasone, dexamethasone, flumethasone, or mixtures thereof.
In another embodiment of the invention, the internal standard is beclomethasone, d3-bupivacaine, d9-bupivacaine, flumethasone, pentylcaine, or mixtures thereof.
In yet another embodiment of the invention, in addition to a corticosteroid, at least one drug agent is present which optionally is quantified. In one embodiment, the drug agent is bupivacaine.
In another embodiment of the invention, the interfering compounds are inorganic salts, organic materials, physiological materials, or combinations thereof. The interfering compounds are removed using liquidxe2x80x94liquid extraction, solid phase extraction, protein precipitation, or a combination thereof, preferably solid phase extraction.
In another embodiment of the invention, the HPLC column is packed with a low acid silica stationary phase. The NH4OAc:MeOH solution has a concentration of 0.5 mM to about 10 mM NH4OAc in MeOH and H2O.
In yet another embodiment of the invention, the MS has a desolvation temperature of about 250xc2x0 C. to about 450xc2x0 C. The MS has a source block temperature of about 80xc2x0 C. to about 150xc2x0 C. The MS has a desolvation gas flow rate of about 400 l/h to about 860 l/h.
In another embodiment of the invention, the method further comprises quantifying the amount of corticosteroid wherein the peak height of the corticosteroid is quantified using a calibration curve. The calibration curved is obtained by plotting data points of known concentrations of corticosteroid versus a peak height ratio of a known amount of corticosteroid/internal standard. Preferably, the calibration curve has from about 4 to about 9 data points.
As used herein, unless otherwise specified, the term xe2x80x9clower limit of quantificationxe2x80x9d or xe2x80x9cLLOQxe2x80x9d means the lowest non-zero amount of corticosteroid detectable by a LC/MS system.
As used herein, unless otherwise specified, the term xe2x80x9cupper limit of quantificationxe2x80x9d or xe2x80x9cULOQxe2x80x9d means the highest amount of corticosteroid detectable by a LC/MS system.
As used herein, unless otherwise specified, the term xe2x80x9cadductxe2x80x9d means a corticosteroid cluster formed by the addition of another molecule or part of a molecule, such as acetate, to a corticosteroid. The cluster may or may not have a charge. The cluster may be formed by any means known in the art including, but not limited to, ionic bonds, covalent bonds, hydrogen bonds, electric forces, and combinations thereof.
As used herein, unless otherwise specified, the term xe2x80x9cdrug agentxe2x80x9d includes, but is not limited to, a substance used, or potentially used, in the diagnosis, treatment, or prevention of a disease or as a component of a medication. The drug agent may optionally be one administered to patients in conjunction with corticosteroids. Optionally, these drug agents may be detected and quantified substantially simultaneously with the corticosteroid.
As used herein, unless otherwise specified, the term xe2x80x9cinterfering compoundxe2x80x9d includes, but is not limited to, compounds within an analytical sample that hinder, obstruct, or impede one of skill in the art from detecting the corticosteroid and/or other drug agents of interest, such as for example, compounds which are structurally similar to a corticosteroid, or compounds which have the same or similar chromatography retention times with the corticosteroid. Optionally, interfering compounds within a sample that may be removed using extraction methodology prior to analyzing the sample.
As used herein, unless otherwise specified, the term xe2x80x9cremovexe2x80x9d means eliminating or reducing the amount of interfering compounds from a sample in an amount sufficient to reduce or avoid unwanted or overlapping peaks or readings within a HPLC or MS chromatogram which affect detection.
As used herein, unless otherwise specified, the term xe2x80x9cLC/MS/MSxe2x80x9d or xe2x80x9cLC/MSxe2x80x9d means either a multiple analytical apparatus having a liquid chromatography and mass spectrometry or separate liquid chromatography and mass spectrometry apparatus used in sequential order, but not necessarily immediately thereafter.
As used herein, unless otherwise specified, the term xe2x80x9csubstantially simultaneouslyxe2x80x9d means, detecting or quantifying at least two compounds within one sample either sequentially or at the same time.
As used herein, unless otherwise specified, the term xe2x80x9cacetate containing solutionxe2x80x9d includes, but is not limited to solutions of organic solvents, non-organic solvents, or both, having acetate ions.